Ion channels are the primary sensors of many physical stimuli such as voltage, lateral stretch, osmolality and temperature. Of these, the fundamental biophysical principles of temperature-sensing and temperature- dependent gating are perhaps the most enigmatic. Despite the fact that many ion channels in the voltage- gated ion channel (VGIC) superfamily are involved in temperature sensing and that many high-resolution structures are now available, a common structural motif or module responsible for this temperature- dependence has not yet been identified. One possibility is that temperature-sensing phenotype is due to convergent evolution and different ion channels have become temperature-sensitive in different ways. According to this line of thinking, unlike a chemical signal, temperature gating may have less to do with a specific structural fold since it is not bound by rules of stereochemistry. The goal of this proposal is to broadly explore the mechanisms of temperature-dependent gating in ion channels using a multi-pronged approach. In specific aim 1, we will apply the newly developed thermodynamic tools and multi-dimensional NMR spectroscopy to thoroughly characterize the biophysical mechanisms that underlie enhanced temperature- sensitive gating in engineered ion channels. We will test the hypothesis that state-dependent change in solvation of side-chains and lipid acyl chains may underlie temperature-dependence in these ion channels. In the specific aim 2, we will explore the temperature-dependence of electromechanical coupling. The goal here is to use rational design approach to test an alternate mechanism of temperature sensing. In this paradigm, the temperature-sensitivity is not due to the sensor itself but due to temperature-dependence of coupling interactions between the voltage-sensor and pore gates. In specific aim 3, we will probe the mechanisms of temperature-dependent gating in a biochemically tractable prokaryotic channel. We have recently identified that the calcium-dependent gating of MthK potassium ion channel is highly temperature-sensitive. Our proposed studies will combine calorimetry, electrophysiology and structural biology with the power of reverse genetics to understand the molecular mechanisms that underlie temperature-dependence in these archeal ion channels. Taken together, the three specific aims will broadly study the mechanisms of temperature-dependent gating in channels of the VGIC superfamily. We expect that this multi-disciplinary approach will shed light on the biophysical mechanisms that underlie exquisite temperature-dependence in many ion channels.